BACKGROUND Measures to prevent assisted reproductive technologies (ART) mix-ups, such as labeling of all labware and double-witnessing protocols, are currently in place in fertility clinics worldwide. Technological solutions for electronic witnessing are also being developed. However, none of these solutions eliminate the risk of identification errors, because gametes and embryos must be transferred between containers several times during an ART cycle. Thus, the objective of this study was to provide a proof of concept for a direct embryo labeling system using silicon-based barcodes.
METHODS Three different types of silicon-based barcodes (A, B and C) were designed and manufactured, and microinjected into the perivitelline space of mouse pronuclear embryos (one to four barcodes per embryo). Embryos were cultured in vitro until the blastocyst stage, and rates of embryo development, retention of the barcodes in the perivitelline space and embryo identification were assessed every 24 h. Release of the barcodes after embryo hatching was also determined. Finally, embryos microinjected with barcodes were frozen and thawed at the 2-cell stage to test the validity of the system after cryopreservation.
In spite of the promising results obtained so far, the approach reported here for direct embryo labeling has some limitations, such as barcode adhesion to the embryo surface after hatching or the need for micromanipulation to label each individual embryo. Current work in our laboratory is focused on overcoming these limitations, and alternative methods of barcode incorporation into oocytes/embryos are being pursued. In particular, modification of the barcode surface aimed at the selective attachment of barcodes to the outer surface of the zona pellucida by either physical or chemical means is being investigated.
RESULTS Barcodes present in the perivitelline space, independently of their type and number, did not affect embryo development rates. The majority of embryos ( over 90%) retained at least one of the microinjected barcodes in their perivitelline space up to the blastocyst stage. Increasing the number of barcodes per embryo resulted in a significant increase in embryo identification rates, but a significant decrease in the barcode release rates after embryo hatching. The highest rates of successful embryo identification (97%) were achieved with the microinjection of four type C barcodes, and were not affected by cryopreservation.
CONCLUSIONS Our results demonstrate the feasibility of a direct embryo labeling system and constitute the starting point in the development of such systems.